Understanding the relationship between the structure, function, and energetics of proteins is one of the central problems in biochemistry today. Electrostatics govern many key biochemical processes, therefore one of the top priorities is to develop a solid understanding of electrostatic effects in proteins. Many computational methods for structure-based calculations of electrostatic energies and pKas have been developed towards this end, but their accuracy, reliability and utility is still limited. They fail most dramatically for groups secluded from bulk solvent in the protein interior or at interfaces between proteins and other molecules. Unfortunately, these are the cases of greatest biological interest. That is where processes that are governed by electrostatics, such as catalysis, redox, H+ and e- conduction, ion selectivity, and recognition and binding of drugs and other molecules, take place. Computational tools for relating structures to energy and function will play an increasingly important role in the post-genomic era. Therefore, experimental data are needed to test and challenge computational methods, as well as to provide the physical insight needed to improve them. Towards this end, Dr. Garcia-Moreno will perform unprecedented studies of the positional dependence of pKas of ionizable groups buried in the interior of 3 proteins: staphylococcal nuclease, RNaseH, and green fluorescent protein. This will entail burial of ionizable groups in the hydrophobic core by mutagenesis, measurement of pKas with equilibrium methods, and determination of structures by crystallography to characterize microenvironments of the buried group. A specific hypothesis that will be evaluated is that buried waters are more prevalent than is currently recognized, and that they hydrate ionizable groups in the interior of proteins very effectively. These experimental studies will answer longstanding questions about the origins, magnitude and meaning of dielectric effects in the protein interior, and Dr. Garcia-Moreno will guide all further refinement and improvement of existing methods for calculation of electrostatic effects in proteins. To the extent that these studies improve our ability to estimate stability and binding affinities from structure, Dr. Garcia-Moreno will have a significant impact on rational and structure-based design of drugs and proteins.